LANCE EHIA NOTE TECHNICAL TECH NOTE U-TRF #24

COT (MAP3K8) Assay UL TRA ULight-p70 S6K (Thr389) Peptide & Europium-anti-phospho-p70 S6K (Thr389) Antibody

Two LANCE Ultra companion products - two convenient sizes!

ULight™ -p70 S6K (Thr389) Peptide: Europium-anti-phospho-p70 S6K (Thr389) • TRF0126-D: 0.5 nmole, 1,000 assay points* Antibody: • TRF0214-D: 10 µg, 1,562 assay points* • TRF0126-M: 5 nmoles, 10,000 assay points* *0.5 pmol/assay point • TRF0214-M: 100 µg, 15,625 assay points* *40 fmol/assay point PEPTIDE MOTIF: FLGFTYVAP RECOGNIZED MOTIF: FLGFTYVAP Synthetic peptide containing the residues surrounding Thr389 of human p70 S6K; phosphorylation site: Thr389. Europium-labeled mouse monoclonal antibody recognizing phospho-Thr389 in human p70 S6K. VALIDATED FOR KINASE: CDK6/CycD3, HGK, IKK␣, IKK⑀, IRAK1, IRAK4, MAP4K2, MINK, MST1, mTOR, NEK1, NEK2, NEK6, NEK7, PEK, , TAOK2

LANCE Ultra Kinase Assays LANCE® Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W-1024 (Eu), with ULight, an innovative small molecular weight acceptor dye with a red-shifted fluorescent emission. In kinase assays, the binding of an Eu-labeled anti- phospho-substrate antibody to the phosphorylated ULight- labeled substrate brings donor and acceptor molecules into close proximity.

After irradiation of the kinase reaction at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The intensity of the light emission is proportional to the level of ULight- substrate phosphorylation.

Development of a COT (MAP3K8) Kinase Assay Additional reagents COT (MAP3K8) Carna # 07-301 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 OptiPlate™-384, white PerkinElmer # 6007299 TopSeal™ -A PerkinElmer # 6005185 Kinase Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 3 mM

MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20.

Note: MnCl2 might not be required for other .

www.perkinelmer.com Suggested procedure • Dilute the COT (MAP3K8) , ATP, inhibitors and ULight- • Stop kinase reactions by adding 5 µL of 40 mM EDTA prepared p70 S6K Peptide in Kinase Buffer. in 1X Detection Buffer (Stop Solution). Leave for 5 min at RT. • Prepare a 4X Detection Mix by diluting the Eu-anti-phospho- • Add 5 µL of Detection Mix (Eu-anti-phospho-p70 S6K Antibody p70 S6K Antibody to 8 nM in 1X LANCE Detection Buffer. at a final concentration of 2 nM). • Add to the wells of a white OptiPlate-384: • Cover with TopSeal-A and incubate for 1 h at RT. 5 µL of COT (MAP3K8) enzyme • Remove TopSeal-A and read signal with the EnVision® 2.5 µL of inhibitor or Kinase Buffer Multilabel Reader in TR-FRET mode (excitation at 320 nm & emission at 665 nm). 2.5 µL of ULight-p70 S6K Peptide/ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). NOTE: Eu-labeled antibodies and EDTA can be premixed before use as a 2X concentrated Stop Solution/Detection mix to minimize • Cover the plate with TopSeal-A and incubate at room the number of liquid handling steps. temperature (RT).

Experiment 1: Enzymatic Time Course Experiment 2: ATP Titration

[COT (MAP 3K8)] (nM) 50000 50000 20 15 40000 40000 10 5 30000 30000 Km app: 13.8 µM

20000 20000

10000 10000 LANCE Signal (665 nm) LANCE Signal (665 nm) 0 0 0 10 20 30 40 50 60 70 80 90 - ∞ -8 -7 -6 -5 -4 -3 Time (min) Log [ATP] (M) COT (MAP3K8) enzyme was incubated at concentrations ranging from 5 to 20 Serial dilutions of ATP ranging from 10 nM to 100 µM were added to 10 nM COT nM with 50 nM ULight-p70 S6K Peptide and 200 µM ATP. Kinase reactions were (MAP3K8) enzyme and 50 nM of ULight-p70 S6K Peptide. Kinase reactions were terminated after 0 to 90 min by the addition of EDTA. terminated after 90 min by the addition of EDTA.

Experiment 3: Enzyme Inhibition Curve Experiment 4: Z’-factor Determination

30000 30000 +15 µMATP IC > 10 µM 25000 50 25000

20000 20000

15000 15000 Z’=0.84 10000 10000 -ATP 5000 5000

LANCE Signal (665 nm) 0 LANCE Signal (665 nm) ∞ 0 - -11 -10 -9 -8 -7 -6 -5 -4 0 4 8 12 16 20 24 Log [Staurosporine] (M) Well #

Serial dilutions of staurosporine ranging from 30 pM to 10 µM (final concentrations COT (MAP3K8) enzyme at 10 nM was incubated with 50 nM ULight-p70 S6K in 1% DMSO) were incubated with 10 nM COT (MAP3K8) enzyme, 50 nM Peptide and 15 µM ATP (final concentrations in 2% DMSO). Kinase reactions ULight-p70 S6K Peptide and 15 µM ATP. Kinase reactions were terminated after were terminated after 90 min by the addition of EDTA. 90 min by the addition of EDTA. Staurosporine does not inhibit COT activity at a concentration of 10 µM, consistent with literature data.

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©2007 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. OptiPlate, TopSeal and ULight are trademarks and EnVision and LANCE are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors.

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